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Preparation method of recombinant NcoI restriction endonuclease

A technology of restriction endonuclease and recombinant bacteria, applied in the field of molecular biology and cell engineering, can solve the problem of difficulty in obtaining a large amount of NcoI protein

Pending Publication Date: 2021-04-16
上海咏科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no literature report on the expression and purification method of NcoI. It is difficult to obtain a large amount of NcoI protein by conventional methods.

Method used

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  • Preparation method of recombinant NcoI restriction endonuclease
  • Preparation method of recombinant NcoI restriction endonuclease
  • Preparation method of recombinant NcoI restriction endonuclease

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Embodiment 1

[0031] In this embodiment, the operation process of the preparation method of the recombinant NcoI restriction endonuclease involved in the present invention is as follows figure 2 shown.

[0032] Step 1, use the prokaryotic expression vector to insert and construct the plasmid expressing NcoIM and the plasmid of NcoI respectively.

[0033] Gene Synthesis The DNA sequence of the gene encoding NcoIM is shown in SEQ ID NO:1, and the DNA sequence of the gene encoding NcoI is shown in SEQ ID NO:2. The NcoI cleavage site was excluded during the DNA sequence synthesis design, and the gene sequence was optimized based on the amino acid sequence (UniProt ID: NcoIM O85488; NcoI O85489). The amino acid sequence of the gene encoding NcoIM is shown in SEQ ID NO:3; the amino acid sequence of the gene encoding NcoI is shown in SEQ ID NO:4.

[0034] NcoIM coding gene (primer pair shown in SEQ ID NO: 5 and shown in SEQ ID NO: 6) and NcoI coding gene (primer pair shown in SEQ ID NO: 7 and s...

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Abstract

The invention provides a preparation method of a recombinant NcoI restriction enzyme, which comprises the following steps: 1, synthesizing NcoIM and NcoI coding genes, and respectively constructing the NcoIM and NcoI coding genes into corresponding vectors to obtain a recombinant vector with the NcoIM gene and an NcoI protein expression plasmid fused with a purification tag; 2, transferring the recombinant vector with the NcoIM gene into escherichia coli to obtain recombinant bacteria with substrate NcoIM expression; 3, preparing a recombinant bacterium competent cell with substrate NcoIM expression; 4, transferring the NcoI protein expression plasmid fused with the purification tag into a recombinant bacterium competent cell with substrate NcoIM expression to obtain positive monoclone, and culturing to express the NcoI restriction endonuclease; and 5, purifying the NcoI restriction endonuclease. Through the method provided by the invention, a large amount of NcoI restriction endonucleases can be obtained, and the method has a wide application prospect.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell engineering, and in particular relates to a preparation method of a recombinant NcoI restriction endonuclease. Background technique [0002] NcoI is a type II restriction endonuclease in Nocardia corallina, which is characterized in that it specifically recognizes the CCATGG, And cut the first C residue after the 5' end of the double strand, such as figure 1 shown. The cleaved fragments have sticky ends, and are highly practical restriction enzyme types in genetics and genetic engineering. As a restriction endonuclease, its recombinant expression in Escherichia coli will seriously inhibit the replication of the host bacterial base genome, affect the proliferation of strains, and fail to express proteins normally. For the expression of restriction endonucleases, the expression method of low background expression strains is often used internationally, such as using BL2(DE3)plysS to exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/70C12N15/66C12N15/62
Inventor 于博文李建辉单永超
Owner 上海咏科生物科技有限公司
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